[DIYbio] Re: A year at -20... does it work?

Hey guys

A year ago I began an experiment to see how I could store things long term in a DIY manner. The results (note: poor controls. Please don't sue me and take all of this as opinion)

-Plasmids are best stored in E coli stabs

-E coli strains are best stored at -20 at 40% glycerol

Simply put, I put 3 things in the freezer for the year: bacteria culture with 10% glycerol, 20% glycerol, and 40% glycerol. I also put in a stab where I just picked a colony and stabbed it into a tube, normal method.

For the 10%, even though there was clear settling of cells I scraped from the top because the culture was frozen. No colonies appeared on the plate. I did the same with 20% and 1 colony appeared. 40% was not frozen so I just took a tiny from the bottom as well when I streaked and got surprising results

Although I used 1/5 of the small pellet at the bottom, I got *significantly* more colonies than I got with streaking out the entire stab. However, nearly 1/3 to 1/2 of these cells no longer held the RFP plasmid even though I plated on amp. On the stab plate, however (same batch of plates), I did not get a single white colony. There were less colonies, but still about 50 colonies.

So, overall, if you wanna store plasmids without an expensive freezer, store them as stab cultures. I can vouch that it works. (however sometimes mutations can occur in the genomes of the cells in stab cultures http://www.ncbi.nlm.nih.gov/pmc/articles/PMC516597/ )

-Koeng